Antigen-carrying microparticles and their use in the indication of humoral or cellular responses

ABSTRACT

The invention concerns the use, in the induction of an immune response, of a synthetic microparticle polymer carrying on the surface one or more covalently bonded proteins capable of carrying one or more epitopes, the densities of the protein(s) on the surface of the microparticles, and their molecular weights, being adjusted so as to direct the immune response to the induction of a humoral and cellular response or to the induction of a largely cellular response. Said microparticles have an average diameter of approximately 0.25 to 1.5 μm.

BACKGROUND OF THE INVENTION

The object of the present invention is microparticles carrying antigenson their surf ace and their use in the induction of humoral or cellularresponses.

More specifically, the invention also relates to microparticles carryinga significant density of antigens on their surface.

The B cells which express immunoglobulin receptors specific for anindividual antigen are highly effective for the presentation of thisantigen (Rock et al. C., J. Exp. Med. (1984) 160; 1102; Hutchings etal., Eur. J. Immunol. (1987) 17:393). For example, specific B cells canpresent tetanus toxin to T cells at antigen concentrations 10⁴ timeslower than those required for the presentation by nonspecific B cells orperipheral blood monocytes (Lanzavecchia, Nature (1985) 314:537).

In addition, in vivo studies with mice deficient in B cells show thatthese cells are required for the activation of T cells of lymphaticganglions (Janeway et al., J Immunol. (1987) 138:2848; Kurt-Jones et al.A.K., J. Immunol. (1987) 140:3773).

Mice deficient in B cells also show reduced responses with respect tospecific CD4+ and CD8+ T cells from tumors, after immunization withFreund's murine leukemia virus (Schultz et al., Science, (1990) 291).

The capacity of B cells to modify and to present the antigen with a viewto recognition by CD4+ helper T cells restricted by the class II majorhistocompatibility complex (MHC) forms the basis of a model for theactivation of the B cells by T cells (Noelle et al., The Faseb Journal(1991) 5:2770).

The recognition of the peptide-class II MHC complex by CD4+ helper Tcells on the surface of the B cells leads to the formation of physicallystable conjugates between the T cells and the B cells (Kupfer et al. S.J., Proc. National Acad. Sci. USA (1986) 83:6080).

This direct recognition results in the proliferation and thedifferentiation of B cells in response to lymphokines such asInterleukin-2, Interleukin-4 or Interleukin-5.

The induction of the antibody response against an antigen requires thepresentation of the antigen by the B cells.

The majority of the studies on antigen presentation have been carriedout using soluble proteins such as tetanus toxoid, lysozyme, hemocyanin(LH). However, most of the antigens to which the immune system isexposed are contained in complex particulate structures such as bacteriaor parasites.

It is well known that cells which are capable of phagocytosis such asthe macrophages can present bacterial antigens to T cells.

However, it is not known whether cells which do not phagocytose, such asB cells, can present complex antigens of significant size.

It has recently been shown that, in vivo, bacterial antigens must be ina soluble form in order to induce an antibody-dependent response by theT cells (Leclerc et al., J. Immunol. (1990) 144:3174; Leclerc et al., J.Immunol. (1991) 147:3545).

However, it seemed advisable to determine also that, in vivo, bacterialprotein antigens are exclusively presented to the T cells by thephagocytic cells and that the B cells cannot modify antigens in particleform.

SUMMARY OF THE INVENTION

According to the present invention, the capacity of macrophages and Bcells to present the same antigen, in soluble and particulate forms, hastherefore been compared.

In particular, protein antigens, such as lysozyme and TNP-KLH, coupledto poly(acrolein) or polystyrene microparticles of a comparable size tothat of a bacterium, have been used.

According to the invention, it has unexpectedly been shown that B cellswhich present TNP-KLH or lysozyme very effectively are incapable ofpresenting these antigens coupled to beads. However, macrophages presentboth forms of the antigens to T cells.

The study of antigen presentation and the induction of the cellularand/or humoral T response is of particular scientific and medicalimportance.

In fact, directing the response towards a purely cellular response or apurely humoral response could allow vaccination against certainpathogens, modification of certain biological dysfunctions and curingcertain pathologies.

For example, such direction would enable the elimination of persistentinfections or the regulation of allergic responses.

In addition, there are two sub-populations of CD4+ T cells, Th1 and Th2,which have different capacities to produce various lymphokines (Mosmann,Cherwinski, Bond, Giedlin and Coffman, J. Immunol., 136, 2348-2357(1986)). The induction of Th1 or Th2 plays a major role in theresistance to bacterial, parasitic or viral infections. Thus, in thecase of murine cutaneous leishmaniasis, the Th1 protect from infectionwhile the Th2 aggravate the disease. In vitro, B lymphocytes optimallystimulate the proliferation of Th2 clones while a strong proliferationof Th1 clones is observed with adherent cells (Gajewski, Pinnas, Wongand Fitch, J. Immunol., 146, 1750-1758 (1991)).

Directing of the antigen towards presentation by the B cells ormacrophages could allow induction of Th1 or Th2 responses.

Various techniques have been developed in the past to achieve a betterimmune response.

The oldest method consists of activating the immune system withadjuvants. Thus, Freund's adjuvant leads to an increased intensity ofthe humoral and cellular responses. However, such adjuvants have majordisadvantages due to their lack of specificity, toxicity, andimmunological side-reactions which may be caused by their lack ofpurity.

The iscomes (immuno-stimulating complexes) are composed of an antigeniccomplex and an adjuvant, QuilA, which is extracted from trees. Theseparticles have a diameter of about 35 nm and are composed of subunits ofabout 12 nm. They lead to the induction of an immune response but moreoften the antigens are encapsulated and thus then released in theexternal medium. In addition, the technique does not allow accuratecontrol of the type of cells presenting these particles, and theseparticles therefore induce a double humoral and cellular response.

Lastly, from a practical standpoint, these particles are difficulty toprepare, lack stability and have significant toxicity.

Liposomes, which have also been tested for use in inducing an immuneresponse, have the same disadvantages as the iscomes.

Biodegradable microparticles such as for example lactic and glutamicacid polymers have also been developed (Aguado and Lambert, Immuno.Biol., 184, 113-125 (1992)). These particles liberate the antigen in asoluble form during their degradation. This liberation enablespresentation of the antigen by different cells and the induction of ahumoral response without the possibility of direction towards aspecifically cellular response.

Particles composed entirely of recombinant proteins have also beensynthesized. Thus, French patent application FR 2.635.532 describesparticles composed of a hybrid protein between HBs antigen and animmunogenic sequence presumed to induce neutralizing antibodies directedagainst the HIV virus.

Particles containing poliomyelitis toxin have also been produced.

These particles have significant disadvantages. Thus, it is verydifficult to insert long sequences into these particles. In addition,they induce as much humoral as cellular response and it is thus notpossible to obtain specifically one or the other.

Polyacrolein or polystyrene particles to which antibodies have beencoupled have already been used for the development of separationtechniques (Rembaum et al., Immunol. (1982) 52:341-351).

However, no use for the preparation of vaccines and in vivo immunizationhas been reported. The beads used have diameters of 20 to 35 nm(polyacrolein) or of 40 to 120 μm (polystyrene).

Polyacrolein particles of 2 μm diameter have also been used for the invitro study of T response stimulation (Ziegler et al., Eur J. Immunol.(1987), 17: 1287-1296). The activity of these beads was not tested invivo.

In all this work, the size of the particles was not considered to be acritical criterion. However, particles of small size (nanoparticles)such as HBs particles could be presented by B lymphocytes. On the otherhand, particles with too large size (greater than 5-10 microns) couldnot be presented by phagocytic cells.

The various solutions proposed in the prior art, on the one hand toinduce a significant immune response and on the other to direct thisresponse specifically towards one of the two response routes, humoral orcellular, are thus not satisfactory.

The invention offers the development of products giving a good immuneresponse with either a cellular or a humoral direction.

According to the invention, it has unexpectedly been found that such aresponse can be induced by using microparticles, of small size andhaving varied antigenic densities.

The present invention particularly relates to synthetic polymermicroparticles carrying on their surface one or more proteins covalentlybonded to the material of the microparticles, said protein or proteinseach carrying one or more epitopes and being present at a density ofbetween 10⁴ and 5.10⁵ molecules/μm² for each of the proteins.

The invention also relates to the characteristics below, consideredalone or in all technically possible combinations:

The coupling of the antigenic proteins or microparticles must becovalent in order to avoid the liberation of the antigen in solubleform.

The microparticles advantageously have an average diameter of betweenabout 0.25 μm and 1.5 μm, and preferentially of about 1 μm so as to beable to be presented to CD4+ T lymphocytes by phagocytic cells but notby B lymphocytes.

Said microparticles are more particularly characterized in that thecovalent bond is formed by reaction between the NH₂ and/or CO groups ofthe proteins and the material making up the microparticle.

Advantageously such bond is created by using a bridging reagent asintermediate, such as for example glutaraldehyde or carbodiimide.However, any other bifunctional reagent able to form such a bond can beused. Such reagents are known, see for example Synthetic polypeptides asantigens, M. H. Von Regensmortel, J. P. Briand, S. Muller and S. Plane1988 (Elsevier). This bond can also be formed without a bridgingreagent.

The material of the microparticle can advantageously be a biocompatiblepolymer, such as an acrylic polymer, for example polyacrolein orpolystyrene or the poly(alpha-hydroxy acids), copolymers of lactic andglycolic acids, or lactic acid polymers.

By polymer should be understood any homopolymer or hetero- orco-polymer.

It must allow covalent bonding of the proteins to the material and mustnot cause a rejection or toxic reaction by the organism into which itmay be injected. Advantageously, for human therapeutic applications, itshould be a biodegradable polymer, for example a polymer able to bedegraded by cells containing lysosomal enzymes, such as the macrophages.

Such biodegradable materials can include lactic and glutamic acidpolymers, starch or polymers used for biomedical applications, and inparticular those used for sutures.

Such a microparticle can carry on its surface, in addition to theantigenic proteins, molecules able to activate the immune system, suchas the interleukins, in particular gamma-interferon or interleukin 4.

These microparticles can carry one or more proteins which can themselveseach contain one or more epitopes. Such proteins can be glycoproteins,synthetic peptides containing an epitope or several epitopes, or anyother nonprotein molecule or molecule containing a protein portion ableto induce an immune response.

The microparticles which are the object of the present invention can inaddition be encapsulated in order to protect the antigens fixed to theirsurfaces from degradation and to transport them to their site of action.

They can thus comprise a nucleus formed from a polysaccharide matrix, towhich are bound the antigens, an initial lipid layer bound covalently tothe nucleus and a second layer of amphophilic molecules.

Another object of the invention is drugs or vaccines comprising themicroparticles described above, as well as pharmaceutical compositionscharacterized in that they contain them, in combination withpharmaceutically compatible diluents or adjuvants.

The present invention generally relates to the use of synthetic polymermicroparticles carrying on their surface one or more covalently bondedproteins, said protein or proteins each carrying one or more T or Bepitopes, for the production of a drug or vaccine for inducing an immuneresponse, according to which the densities of the protein or proteins onthe microparticle surfaces are adjusted so as to direct said immuneresponse towards a largely humoral response or a largely cellularresponse.

Another aspect of the invention has as object a process for theproduction of a drug or vaccine whose immune response is either largelyhumoral or largely cellular, of Th1 or Th2 type, said process beingcharacterized in that at least one protein carrying one or more epitopesis covalently fixed to synthetic polymer microparticles or beads, thedensity of the protein fixed to the surface being varied according tothe type of response required.

In order to induce a cellular and humoral response, microparticlesshould preferentially be used with a density for each of the proteinscarrying an epitope of a minimum of 10⁵ and preferentially of about5.10⁵ molecules/μm². Such densities approximately correspond for a beadof 1 μm diameter to quantities of protein on the microparticle surfaceof 10⁵ and 4.10⁵ molecules respectively.

For the induction of a largely cellular response, restricted class IICD4+, microparticles should preferentially be used with a density foreach of the proteins carrying an epitope of between 10⁴ and 5.10⁴protein molecules/μm².

In order to encourage the induction of this cellular response,microparticles should preferentially be used carrying on their surfaceproteins with molecular weights greater than 50 kD.

The other characteristics of these microparticles are those given abovefor the high-density microparticles.

The proteins and antigens covalently bonded to the microparticles dependon the anticipated application for said microparticles.

They also depend on the type of immune response required, but also onthe disease or ailment to be treated or against which the patient is tobe protected.

Examples of epitopes which may be used are the epitopes from the Pre S2region of the HBS antigen of the viral hepatitis virus, with thefollowing sequences:

T epitope: Pre S:T (120-132)

MQWNSTTFHQTLQ (SEQ ID NO:1)

B epitope: Pre S:B (132-145)

QDPRVRGLYFPAGG (SEQ ID NO:2)

Other examples are the epitopes of the VP1 protein of the poliomyelitisvirus whose sequences are as follows:

T epitope: C3:T (103-115)

KLFAVWKITYKDT (SEQ ID NO:3)

B epitope: C3:B (93-103)

DNPASTTNKDK (SEQ ID NO:4)

Another example is the epitope of the V3 loop of the GP120 protein ofthe HIV1 virus whose sequence is the following:

T+B epitope: V3 loop

INCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNI (SEQ ID NO:5)

The B epitopes may preferentially be used to induce a humoral responseusing high-density microparticles and the T epitopes to induce a largelycellular response using microparticles with a low density of surfaceproteins.

Such microparticles may be injected into patients who are to be treatedin a therapeutic or prophylactic manner in ways known to those skilledin the art, for example by subcutaneous, intra-peritoneal, orintravenous injection or by any other means for inducing an immuneresponse.

This subject is discussed in Current protocols in immunology (edited byJ. F. Coligen, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober, published by Wiley-Interscience), in which the range ofimmunological techniques is listed.

A particular advantage of the present invention rests in the fact thatit enables the induction of a humoral or cellular immune responsewithout the addition of adjuvants to the beads or microparticles.However, the addition of nontoxic adjuvants not causing an immuneside-reaction is also envisageable within the scope of use according tothe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated without being in any way restrictedby the following examples in which:

The diagrams of FIG. 1 are the results of fluorimetric analyses (FACS)of microparticles carrying the KLH or TNP-KLH antigens. The ordinatesshow the antibody used (PBS-control, anti-KLH, anti-TNP). The abscissaeshow the type of microparticle tested: B (KLH), B (TNP-KLH), B (OVA) andB (OVA-TNP) which correspond to the microparticles on which are bondedrespectively KLH, TNP-KLH, ovalbumin and ovalbumin-TNP.

FIGS. 2A to 2D show the capacities of spleen and macrophage cells and ofTNP-specific B cells activated by LPS to present KLH, TNP-KLH,microparticles carrying KLH and microparticles carrying TNP-KLH.

FIG. 3 is a curve showing the proliferative responses of ganglion cellsfrom mice immunized with soluble lysozyme and stimulated in vitro bysoluble lysozyme (FIG. 3A) or by microparticles carrying lysozyme andhaving diameters of 0.25, 0.75 and 1.5 (FIG. 3B). The cell proliferationwas measured by the incorporation of thymidine (CPM) as ordinate, withthe microparticle dilution shown on the abscissa.

FIG. 4 shows the production of IL2/IL4 by a lysozyme-specific hybridomaafter stimulation by soluble lysozyme (FIG. 4A) or by microparticlescarrying lysozyme (FIG. 4B). The microparticle concentrations are shownon the abscissae while the proliferation is shown on the ordinates.

FIGS. 5A and 5B show the activation of the lysozyme-specific T hybridomameasured by the production of IL2/IL4 after stimulation by solublelysozyme (FIG. 5A) or lysozyme coupled to microparticles (FIG. 5B) inthe presence of splenocytes or A20 B cells as cells presenting theantigen.

FIGS. 6A and 6B show the in vitro proliferation after stimulation bysoluble lysozyme of inguinal ganglion cells from mice immunized withrespectively soluble lysozyme in complete Freund's adjuvant (FIG. 6A)and by lysozyme coupled to microparticles (FIG. 6B).

FIGS. 7A and 7B show the in vitro proliferation after stimulation bylysozyme of inguinal ganglion cells from mice immunized by differentconcentrations of soluble lysozyme (7A) or lysozyme coupled tomicroparticles (7B).

FIG. 8 shows the proliferation of ganglion cells from mice immunizedwith lysozyme in complete Freund's adjuvant or in PBS or in the presenceof microparticles coupled to patella hemocyanin, or KLH (Keyhole LimpetHemocyanin).

FIG. 9 shows the proliferative response of ganglion cells from miceimmunized with soluble hemoglobin in adjuvant (Hb+CFA) or coupled tobeads (B-Hb).

FIG. 10 shows the proliferative response of mouse cells stimulated bysoluble ovalbumin (OVA+CFA) or ovalbumin coupled to beads (B-OVA).

The proliferative response of the hemoglobin and ovalbumin respectivelywas measured as ordinate by the incorporated radioactivity (CPM) as afunction of the quantity of hemoglobin or ovalbumin (as abscissa) usedfor restimulating the cells.

FIG. 11 illustrates the proliferation of mouse cells after immunizationwith the C3 peptide as its soluble form (C3:T+CFA) or as microparticles(B-C3: T). The cells were restimulated in the presence of a quantity ofsoluble C3 (as abscissa) and the proliferation was measured (ordinate).

FIG. 12 shows the proliferation of mouse cells after immunization withthe pre-S:TB peptide as its soluble form (Pre-S:TB+alum) or asmicroparticles (B-pre-S:TB) or the pre-S:B peptide in particulate form(B-Pre S:B) and restimulated by the Pre-S peptide.

FIGS. 13A and 13B represent respectively the levels of anti-lysozymeantibody (FIG. 13A) and anti-KLH antibody (FIG. 13B) of mice immunizedwith lysozyme and alum adjuvant, with microparticles carrying lysozymeor with microparticles carrying LH.

FIGS. 14 and 15 show the antibody response of mice immunized withhemoglobin (FIG. 14) or ovalbumin (FIG. 15) in soluble or particulateform. The log of the antibody titer is shown on the ordinate with timeon the abscissa.

FIG. 16 shows the antibody response of mice immunized by soluble pre-S:TB peptide, or the pre-S: TB or pre-S: B peptides in particulate forms.The ordinate and the abscissa of this curve are as defined for FIGS. 14and 15.

FIG. 17A shows the proliferation of mouse cells after immunization byinjection of lysozyme with Freund's adjuvant, after in vitro stimulationby microparticles carrying different densities of lysozyme.

FIG. 17B shows the in vitro proliferation of mouse cells afterimmunization in vivo with lysozyme or with beads carrying lysozyme afterstimulation by different concentrations of lysozyme.

FIG. 18 is a diagram illustrating the production of anti-lysozymeantibody in cells from mice immunized by injection of lysozyme andFreund's adjuvant or by lysozyme-carrying microparticles.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1

Preparation of beads coupled to KLH or to ovalbumin.

1. Materials and methods and presentation by B cells or by macrophages.

The mice were 6 to 8 week old BALB/c and DBA/2 females.

The antigens were KLH and ovalbumin (OVA) marketed by Sigma Chemical (StLouis, USA). The trinitrophenylated hemocyanin (TNP4-KLH) was preparedas previously described (Shutze et al., J. Immunol. (1989) 142:2635).

1.1 Covalent coupling of antigens to poly(acrolein) microparticles.

Poly(acrolein) microparticles with diameter between 0.25 and 1.5 μm,marketed by Polysciences Inc. (Washington Pa.) were coupled to ovalbuminor KLH as previously described (Rembaum et al., Immunol. (1982) 52:341;Ziegler et al., Eur. J. Immunol. (1987) 17:1287).

1 ml of these microparticles was washed twice in PBS and resuspended in1 ml of KLH or ovalbumin (5 mg/ml in PBS). After 3 hours' incubation atambient temperature, the microparticles were washed twice in PBS andresuspended in 2 ml of PBS containing 15 of bovine serum albumin (BSA)and antibiotics. The microparticles thus obtained were stored at 4° C.until used.

The microparticles carrying the TNP-OVA or TNP-KLH antigens wereprepared by incubation of microparticles carrying OVA or KLH with TNBS(trinitrobenzene sulfonate).

2 ml of the microparticles which had been coupled to KLH or ovalbuminwere washed twice in PBS and resuspended in 2 ml of cacodylate buffercontaining 10 mg/ml of TNBS. The microparticles were incubated for 30minutes in darkness at ambient temperature and washed three times inPBS. They were resuspended in 2 ml of PBS containing 1% BSA andantibiotics and stored at 4° C.

1.2 Analysis by flow cytofluorimetry.

50 μl of microparticles were washed twice in PBS containing 1% of BSAand incubated for 40 minutes at 4° C. with mouse anti-KLH or anti-TNPserum. After two washes the microparticles were incubated with goatantibody coupled to FITC (fluoroisothiocyanate) directed against mouseimmunoglobulins (Biosys, Compiegne, France) for 40 minutes at 4° C.

After four washes the microparticles were resuspended in 1 ml of PBScontaining 1% of BSA.

The fluorescence intensity was measured by use of a FACSAN flowcytometer (Becton Dickinson, Mountain View, Calif.).

1.3 Culture medium.

The lymphocytes were cultured in RPMI 1640 (Seromed, Munich, FRG)complemented with 2 mM L-glutamine, 10% of FCS (fetal calf serum)inactivated by heat, 50 μM of 2-ME and antibiotics.

1.4 Establishment of the KLH-specific T cell line.

This cell line was established and maintained according to the methoddescribed by Taylor et al. (IRL Press, New York) and Galelli et al. (J.Immunol. (1990) 145:2397).

Inguinal ganglion cells (4 10⁶ /ml) from DBA/2 mice which 8 days beforeremoval of the cells had received an injection at the base of the tailof 100 μg of KLH in emulsion in complete Freund's adjuvant were culturedfor 4 days in the culture medium in the presence of KLH (100 μg/ml).

The cultures were incubated in a humid atmosphere under 7.5% of CO₂ at37° C.

A cell line was established from this initial culture by serial passageof T cells purified on Ficoll (2.10⁵ /ml) in the presence of DBA/2 mousespleen cells which had been irradiated (3000 rads) for 6 to 8 days (restperiod) or with irradiated spleen cells plus KLH (100 μg/ml) for 4 days(stimulation period).

The T cells used in these experiments were collected 8 to 10 days aftertheir last contact with KLH.

1.5 Estimation of the T cell proliferation.

Cultures in triplicate containing 5.10⁴ T cells purified on Ficoll, and5.10⁴ purified and irradiated (900 rad) TNP-specific memory B cells, or5.10⁵ irradiated (3000 rad) entire spleen cells, or 10⁵ irradiated (3300rad) adherent spleen cells, or 10⁵ irradiated (3300 rad) A20 B celllymphoma cells positive for class II MHC (Kim et al., J. Immunol. (1979)122:549), or 10⁵ TNP-specific virgin B cells activated by LPS as sourceof the cells presenting the antigens, and different concentrations ofantigen were incubated in flat-bottomed microculture plates (Corning,Cambridge, Mass.) under a total volume of 0.2 ml/well of completemedium. The T cell proliferation was estimated by incorporation oftritiated thymidine during the final eight hours of 3 days' culture.

The results are expressed as the geometric mean of three cultures, afterelimination of background noise. The standard deviation was less than15% of the mean.

1.6 TNP-Specific B cells.

The TNP-specific B cells from normal mice were purified by adsorptionand elution on TNP8-gelatin according to the method described by Haasand Layton J. E., J. Exp. Med. (1975) 141:1004.

This method was modified in order to obtain populations enriched inTNP-specific memory B cells from spleens from previously immunized mice,as described previously (Galelli et al., J. Immunol. (1990) 145:2397)).The TNP-specific memory B cells were selected on the gelatin carrying ahapten (TNP2-gelatin), by testing the affinity of TNP receptors bycomparison with virgin B cells, and the capacity to secrete largequantities of anti-TNP immunoglobulin G in the presence of low antigenconcentrations.

10⁸ spleen cells containing neither erythrocytes nor dead cells weresuspended in 3 ml of HEPES (50 mM) buffered with DMEM (Seromed, Munich,Germany) and incubated in plastic Petri dishes covered withTNP2-gelatin. The dishes were gently agitated for 15 minutes at 4° C.,then washed 10 times with DMEM at ice temperature. The adherent cellswere eluted by the addition of 5 ml of DMEM reheated to 37° C. and thebonded TNP-gelatin was eliminated by digestion with collagenase (CLSIIICollagenase from Worthington Biochemicals, Freehold, N.J., 100 U/ml) for15 minutes at 37° C.

This method gives an overall yield, expressed as a percentage of theoriginal number of spleen cells, of 0.3 to 0.6% of cells bonding to TNPfrom the immunized mouse spleen. The cells were cultured overnight,before the addition of other cells and reagents, in order to enable thereexpression of surface immunoglobulins modified by the treatment withthe collagenase. The presence of free TNP receptors on the cells wasevaluated from their capacity to bind erythrocytes carrying TNP on theirsurface.

55 to 76% of the cells obtained from the immunized mice formed rosetteswith the mouse spleen B cells modified by the TNP. These cells did notproliferate in response to concanavalin A but were 20 times enriched,for the cells which secreted anti-TNP immunoglobulin G after stimulationby TNP-LH, by comparison with non-fractionated spleen cells.

1.7 TNP-Specific virgin B cells activated by LPS.

TNP-Specific virgin B cells from non-immunized mice were purified byadsorption and elution on TNP8 gelatine as described previously. Thesecells were cultured to a density of 2.10⁶ per ml in a medium containing50 μg/ml of LPS (Salmonella enteriditis, Difco Laboratories, Detroit,Mich.) for 3 days. The non-adherent lymphoblasts were purified by use ofFicolle-Hypaque (Pharmacia, Piscataway, N.J.), then washed and used assecondary cells.

1.8 Macrophages.

The macrophages were obtained from non-immunized spleen cells byadhesion for 4 hours at 37° C. followed by washing of the cells in orderto eliminate the non-adherent cells as previously described (Kakiochi etal., J. Immunol. (1983) 131:109).

2. Results.

2.1 Verification of antigen coupling to the microparticles.

The KLH was covalently bonded to polyacrolein microparticles withdiameter between 0.25 and 1.5 μm. The coupling of the KLH to themicroparticles was checked by flow cytofluorimetric analysis usinganti-KLH mouse serum.

The results obtained with 1.5 μm microparticles are shown in FIG. 1.

The 1.5 μm microparticles were coupled to ovalbumin (B OVA) or KLH(B-KLH). The TNP-OVA or TNP-KLH microparticles (designated respectivelyB(TNP-OVA) and B(TNP-KLH)) were prepared by incubation of microparticlescarrying OVA or KLH with TNBS. The cytofluorimetric analysis was carriedout on microparticles incubated in the presence of PBS or anti-KLH oranti-TNP mouse serum. After washing, the microparticles were incubatedwith goat antibodies bonded to FITC directed against mouseimmunoglobulins and were analyzed by flow cytometry.

Similar results were obtained with 0.25 and 0.75 μm microparticles.

Control microparticles coupled to ovalbumin were not recognized by theanti-KLH serum.

2.2 Comparison of the ability of different splenocyte populations topresent soluble or particulate antigens.

The ability of non-fractionated splenocytes, macrophages, andTNP-specific virgin B cells was compared for presentation of soluble orparticulate KLH and TNP-KLH to KLH-specific T cells.

In these experiments the splenocyte populations were prepared fromnon-immunized mice. After purification, the TNP-specific B cells wereactivated for three days by LPS; it is known that the lymphoblastsinduced by LPS are extremely efficient for antigen presentation(Kakiochi et al., J. Immunol. (1983) 131:109).

The results are illustrated in FIG. 2 for which 5.10⁸ irradiatedsplenocytes, 10⁵ adherent cells or 10⁵ TNP-specific virgin B cellsactivated by LPS were cultured with 5.10⁴ KLH-specific T cells in thepresence of different quantities of soluble KLH (A), soluble TNP-KLH(B), or fixed on microparticles (B KLH) (C), or (B TNP-KLH) (D). The Tcell proliferation was estimated on day 3.

As shown in FIG. 2 (2A and 2B) the macrophages and the B cells activatedby LPS efficiently stimulated the T cells when they were incubated withsoluble KLH or TNP-LH.

In contrast to these results, only the macrophages, and not theLPS-activated TNP-specific B cells, were able to stimulate theKLH-specific T cells (FIGS. 2C and D) when the microparticles carryingKLH or TNP-KLH were used.

These results show that the macrophages are responsible for the activityof spleen cell antigen presentation when particulate antigens are used.

The inability of the TNP-specific B cells to present the particulateantigen has thus been demonstrated.

EXAMPLE 2

Induction of a lysozyme specific CD4+ T-proliferative response in vivoand in vitro by lysozyme-coupled microparticles.

1. MATERIALS AND METHODS.

1.1 Antigens.

The lysozyme (LYSO) and the Limulus hemocyanin (LH) were from SigmaLaboratories.

1.2 Coupling of the antigen to the microparticles.

The soluble antigen was made particulate by coupling to microparticles(Polysciences) of between 0.2 and 1 μm diameter. Two coupling methodswere used:

1.2 a) Direct covalent coupling without activating agent.

The polyacrolein beads or microparticles possess aldehyde groups capableof spontaneous reaction with the amine functions of the proteins.

1 ml of beads were washed 4 times in PBS and then taken up in 1 ml ofantigen at 5 mg/ml concentration. After 3 hours' incubation at ambienttemperature, the beads were washed 3 times in PBS and incubated for 30minutes in 1 ml of PBS-1% human albumin in order to saturate the freereactive groups on the beads. After washing, the particles were thentaken up in 2 ml of PBS-1% human albumin-1% antibiotic and stored at +4°C.

b) Covalent coupling by glutaraldehyde.

The antigen was coupled to polystyrene beads by glutaraldehyde, whichwas capable of forming a Schiff's base with the protein amine groups.

0.5 ml of beads were washed 3 times in PBS and taken up in 0.5 ml of 8%glutaraldehyde. After 6 hours' incubation at ambient temperature, thebeads were washed twice and then taken up in 1 ml of antigen atconcentration 400 μg/ml. After incubation overnight at ambienttemperature, the beads were washed and incubated with 1 ml of 0.2Methanolamlne for 30 minutes in order to block the free aldehydefunctions of the glutaraldehyde.

After a final washing, the particles were taken up in 1 ml of PBS-1%human albumin-1% antibiotic then stored at +4° C.

This coupling method enabled the quantity of proteins coupled to themicroparticles to be determined by spectrophotometry. The absorbances ofthe 400 μg/ml protein solution and the supernatant obtained afterincubation of the beads with this protein solution were measured at 280nm. Given the number of beads used for the coupling, the differencebetween the quantity of protein before coupling and the residualquantity after coupling could be used to estimate the quantity oflysozyme coupled per particle.

1.3 Immunization protocol.

BALB/c females, haplotype h-2^(d), aged 6 to 9 weeks (reared in theInstitut Pasteur) were used.

immunization by intra-peritoneal route: 100 μg of lysozyme with 1 mg ofalum were injected, or different quantities of antigen coupled to beadswithout adjuvant,

immunization by subcutaneous route: 100 μg of lysozyme in emulsion withcomplete Freund's adjuvant were injected at the base of the tail, ordifferent quantities of antigen coupled to beads.

The serum of each mouse was sampled 7 to 14 days after injection. Theantibody strength of the serum was measured by the ELISA assay.

The cell proliferative response was measured on inguinal ganglionsand/or on the spleen, sampled 7 and/or 14 days after each injection.

1.4 Detection of antibodies by ELISA.

The antigen (lysozyme) was incubated at a concentration of 5 μg/ml in 50mM pH 9.6 carbonate buffer in the microplates (Nunc) for one night at 4°C. After washing with a 0.01% PBS-Tween 20 buffer, the different serumdilutions to be tested, in 1% BSA buffer, were incubated for 1 hour at37° C. After washing, 100 μl of a mouse anti-Ig conjugate (completeanti-Ig supplied by Diagnostics Pasteur and specific anti-Ig by Sigma)were placed in each well, marked with goat peroxidase; this wasincubated for 1 hour at 37° C. After washing, a substrate solution wasadded freshly prepared as follows: 0.5 mg/ml of orthophenylenediamine(Sigma) in a 0.1M citric acid-0.2M disodium phosphate buffer, pH 5, towhich was added H₂ O₂ to 1/2500.

A yellow coloration revealed the presence of specific antibodies; theenzyme reaction was stopped 8 minutes later by the addition of 50 μl of11.5% H₂ SO₄.

The absorbance of each well was measured at 492 nm by an optical densityreader (Dynatech). The negative control was made with 1:100 serum fromnon-immunized BALB/c mice. The results are expressed either in OD×1000from measured absorbance, corrected for the absorbance in absence ofserum, or by the antibody titer calculated from the linear regressionbased on the absorbance obtained with the serum from the non-immunizedBALB/c mice.

When the antigen was in particulate form, the ELISA assay was carriedout in tubes. The serum dilutions to be tested were incubated directlywith the antigen coupled to the beads (8.10⁸ particles/ml).

Washings were made by centrifuging in 0.1% PBS-Tween 20 buffer. When theenzyme reaction had finished, 200 μl from each tube was transferred ontoa microplate and the absorbance then measured.

1.5 Inhibition of the fixation of the anti-lysozyme antibody by theELISA assay.

The ELISA assay measured the fixation of specific antibodies present inthe serum of the immunized BALB/c mice by the lysozyme. This fixationwas reduced if the serum was preincubated (before the ELISA assay) withthe antigen: soluble lysozyme or lysozyme coupled to beads, which thenbehaved as an inhibitor.

The anti-lysozyme serum was preincubated with soluble lysozyme orlysozyme coupled to beads for 1 hour at 37° C., then for 1 night at 4°C.; the reaction was carried out in the tubes. The fixation ofantibodies not bonded to the inhibitor was evaluated by the ELISA assay(triplicates) on microplates, in which the wells were covered with 5μg/ml of lysozyme. The absorbance of each well was measured at 492 nm,and corrected for the absorbance in the absence of serum. The negativecontrol was carried out with 1:100 serum from non-immunized BALB/c mice.The absorbance without inhibitor during the preincubation of the serumcorresponded to the maximum anti-lysozyme antibody fixation.

Results are expressed as a percentage of the inhibition of the antibodyfixation and calculated according to the ratio: ##EQU1##

The graphical representation of the soluble lysozyme concentrationnecessary for 50% inhibition, together with the number of beads coupledto lysozyme, enabled estimation of the quantity of lysozyme fixed perparticle.

1.6 Stimulation of a lysozyme-specific T hybridoma

A T hybridoma was produced by immunization of BALB/c mice with lysozyme.It specifically recognized peptide 108-116 of lysozyme, in combinationwith molecules of the class II I-E^(d) Major Histocompatibility Complex.

10⁵ T hybridoma cells were stimulated by increasing antigenconcentrations: lysozyme or coupled beads, in the presence of differentcells presenting the antigen: 5.10⁵ irradiated splenocytes (3000 rad) ofBALB/c mice or 10⁵ cells of B lymphoma A20, restricted by Class II MHCmolecules. The cells were cultured (in triplicate) in a complete RPMImedium (SEROMED) supplemented with 10% decomplemented fetal calf serum,50 μM β-mercaptoethanol, 2 mM glutamine, 100 UI/ml penicillin and 100μg/ml streptomycin, on flat-bottomed microplates (Corning 25860). Thepositive control was performed by stimulation of the hybridoma by the Tlymphocyte mitogen:concanavalin A at 5 μg/ml.

The supernatant was removed after 24 h culture at 37° C. (7.5% CO₂),then frozen to -20° C. for a minimum of 16 h. The stimulation of thehybridoma was measured by the IL2 concentration of the supernatant in aCTL-L cell proliferation test. Standard deviations have not been givenas the error was lower than 10% of the mean of the triplicates.

1.7 Determination of IL2 and IL4

The CTL-L line is dependent on Interleukin 2 and Interleukin 4; it wasmaintained in culture in complete medium enriched with 20% of ratsplenocyte supernatant, incubated 36 h with 2.5 μg/ml of concanavalin A.

After thawing, the culture supernatants (tested 1/2) were incubated inthe presence of 2.25.10⁴ CTL-L cells, previously washed three times inRPMI 1640 medium, for 3 days at 37° C. (7.5% CO₂).

The cell proliferation was measured by the addition of tritiatedthymidine with specific activity 1 Ci/mmole, at a level of 2 μCi/ml ofculture, for the last 16 hours of culture.

The cell DNA was recovered after cell lysis and filtration using a"Skatron". Radioactivity incorporation was counted by scintillationusing a beta counter.

The results are expressed in cpm based on the mean of the triplicates,corrected for the radioactivity incorporated in the absence of antigen.

1.8 Proliferation test

The spleen and/or the inguinal ganglions were removed under sterileconditions 7 or 14 days after immunization of the mice (see immunizationprotocol). 8.10⁵ Cells were incubated in the presence of differentconcentrations of antigen, soluble or coupled to beads. The cells werecultured (in triplicate) in RPMI 1640 medium (SEROMED) supplemented with1.5% decomplemented fetal calf serum, 0.5% normal mouse serum, 50 μMβ-mercaptoethanol, 2 mM glutamine, 100 UI/ml penicillin and 100 μg/mlstreptomycin, on microplates (Corning 25860) for 4 days at 37° C. (7.5%CO₂).

The cell proliferation was measured by the incorporation of tritiatedthymidine with specific activity 25 Ci/mmole, at a level of 2 μCi/ml ofculture, for the last 16 hours of culture. The cell DNA was recoveredafter cell lysis and filtration using a Skatron. Radioactivityincorporation was counted by scintillation using a beta counter.

The results are expressed in cpm based on the mean of the triplicates,corrected for the radioactivity incorporated in the absence of antigen.

2--RESULTS.

2.1. Stimulation of ganglion cells from mice immunized with lysozyme bylysozyme coupled to microparticles.

In the tests illustrated by FIGS. 3A and 3B, BALB/c mice were immunizedby subcutaneous injection at the base of the tail of soluble lysozymecomplemented with Freund's adjuvant (CFA).

After 14 days, the inguinal ganglions were removed, and theproliferative response of these cells was tested in vitro againstdifferent concentrations of lysozyme or against different concentrationsof microparticles coupled to lysozyme. The results are expressed in cpmcorrected for the value obtained without antigen.

Soluble lysozyme induced substantial proliferation of cells from miceimmunized by this antigen in Freund's adjuvant (3A). The in vitrostimulation of these cells by lysozyme-microparticles revealed that thelatter are able to induce a very strong cell proliferation (FIG. 3B).The microparticles with very large diameter, 0.81 and 0.96 μm(spontaneous coupling), were very effective.

2.2. Stimulation of T hybridoma by lysozyme coupled to beads

FIGS. 4A and 4B correspond to the results for stimulation oflysozyme-specific T hybridoma by soluble lysozyme (4A) or lysozymecoupled to microparticles (4B). The degree of stimulation of thehybridoma was measured by the level of IL-2/IL-4 produced.

In the presence of irradiated splenocytes, the T hybridoma was stronglystimulated by soluble lysozyme (FIG. 4A). In the presence of thesecells, the large lysozyme-microparticles (0.81 and 0.96 μm) also causedsubstantial production of IL-2/IL-4 (FIG. 4B), in contrast to the 0.5and 0.25 μm microparticles which were not able to stimulate the specificT hybridoma.

2.3. Inability of B lymphoma A20 cells to present lysozyme coupled tobeads to lysozyme-specific T hybridoma.

It is known that B cell tumors carrying Ia receptors can be used asantigen-presenting cells for antigens which do not react with the Igreceptor but which are fixed by B cell tumors by nonspecific mechanisms(Walker et al., J. Immunol. (1982) 128:2164; Glimcher et al. J. Exp. Med(1981) 155:445; MacKean et al. J. Exp. Med. (1981) 154:1419).

The capacity of one of these B cell tumors, the A20 line, to presentlysozyme in soluble or particulate form was thus tested.

The presentation of soluble or particulate lysozyme was compared usingtwo sources of antigen-presenting cells: either a heterogenous source,irradiated entire splenocytes, or B cells from the A20 lymphoma. Whenthe antigen was in soluble form (FIG. 5A), it could stimulate the Thybridoma equally well in the presence of splenocytes as of A20 B cells.However, particulate lysozyme was presented only by splenocytes and notby A20 B cells (FIG. 5B).

These results confirm that splenocytes can present an antigen to Tcells, either in soluble or particulate form. However, B lymphocyteswere not able to present an antigen rendered particulate by coupling toa bead of a size of the order of a micron.

2.4 Induction of T proliferative responses by injection of lysozymecoupled to microparticles to mice.

The in vivo immunogenicity of the antigen coupled to microparticles wasanalyzed by immunizing BALB/c mice with lysozyme in complete Freund'sadjuvant or with this antigen coupled to polyacrolein beads. After 14days, cells from draining ganglions of these animals were stimulated invitro by different concentrations of soluble lysozyme.

In the presence of soluble lysozyme, the ganglion cells proliferatedstrongly, whether originating from mice immunized with soluble lysozymeor lysozyme-microparticles (FIG. 6A). This shows that in both caseslysozyme-specific T cells were sensitized in vivo. After injection ofLH-microparticles to mice, representing the specificity control, theganglion cells of these animals were not able to proliferate in responseto stimulation by soluble lysozyme in vitro (FIG. 6B). The cellularresponse in vivo is thus specific to the protein antigen coupled tomicroparticles, used during immunization of the mice.

The proliferative response of the cells sensitized by 10⁹lysozyme-microparticles (corresponding to 1 μg of lysozyme), in theabsence of adjuvant, was as high as that of cells from animals immunizedwith 100 μg of lysozyme in Freund's adjuvant (CFA) (FIG. 6A). In orderto confirm and clarify this result, proliferative responses of ganglioncells from animals having received different doses of lysozyme in CFA ordifferent concentrations of coupled microparticles were compared, afterin vitro stimulation by soluble lysozyme.

In the case of FIGS. 7A and 7B, the mice had been immunized bysubcutaneous injection at the base of the tail of soluble lysozyme andcomplete Freund's adjuvant (CFA) (FIG. 7A) or beads coupled to antigenwithout adjuvant (FIG. 7B).

After 14 days, the inguinal ganglions were removed, and theproliferative response of these cells was tested in vitro againstdifferent lysozyme concentrations. The results are expressed in cpmcorrected for the value obtained without antigen.

In FIG. 7B, it should be noted that the designations 10⁹, 10⁸, 10⁷ and10⁶ B-LYSO correspond respectively to weights of 1; 0.1; 0.01 and 0.001μg of lysozyme.

These results show that the ganglion cells from animals immunized withlysozyme-carrying microparticles proliferate in vitro after contact withlysozyme, thus demonstrating sensitization of the T cells specific forthis antigen.

Comparison of the concentration effects (FIG. 7) shows that 1 μg oflysozyme coupled to beads gives a response quasi-equivalent to that of 1μg of antigen injected in CFA.

FIG. 8 represents the proliferative response of cells from miceimmunized with lysozyme in complete Freund's adjuvant (CFA) or in PBSwith microparticles coupled to LH. The addition of LH beads to lysozymedid not lead to induction of high proliferative responses, which showsthat the lysozyme must be covalently coupled to the microparticles toinduce T-proliferative responses.

2.5--Induction of T-proliferative responses by injection of mice withhemoglobin or ovalbumin coupled to microparticles

Mice were immunized with hemoglobin or ovalbumin in complete Freund'sadjuvant, or with these proteins covalently coupled to the same type ofparticles as in the previous examples (polystyrene, 1 μm diameter).

The ganglion cells from these animals were restimulated in vitro by thesoluble proteins and the cell proliferation was measured.

The results obtained for hemoglobin (Hb) are shown in FIG. 9, while FIG.10 shows the results obtained for ovalbumin (OVA).

These results overall show that these proteins coupled to microparticlesare able to sensitize CD4+ T lymphocytes specific to these proteins invivo, in the absence of adjuvant.

2.6--Induction of T-proliferative responses by injection of syntheticpeptides

2.6.1--T Epitope from region C3 of the VP1 protein The T epitope of theC3 region (C3: T, 103-115) of the poliovirus protein was synthesized andcovalently coupled to 1 μm beads. These beads were injected into BALB/Cmice.

The results in FIG. 11 clearly show that the T epitope coupled to thebeads (B-C3:T) induced a strong T-proliferative response for quantitiesof the order of 10⁹ beads injected per mouse.

2.6.2--Pre-S:T peptide of the HBS antigen

The pre-S:T peptide (120-132) of the HBS antigen was synthesized andcovalently coupled by glutaraldehyde to beads of 1 μm diameter.

FIG. 12 shows that the injection of 10⁹ beads to DBA/1 mice induced astrong T-proliferative response, stronger than that obtained with thepeptide in CFA. The injection of beads not containing the B epitope didnot induce a proliferative response, showing the specificity of theresponse.

EXAMPLE 3

Induction of antibody response by microparticles carrying an antigen.

The materials and methods were similar to those of Example 2.

1. Lysozyme or Limulus hemocyanin

For FIGS. 13A and 13B, BALB/c mice were immunized by intra-peritonealinjection with 100 μg of soluble lysozyme in adjuvant (alum) or withbeads coupled to antigen: lysozyme or Limulus hemocyanin (LH), withoutadjuvant.

The injections were carried out at D0, D21, D42, the serums were takenat D20, D31, D40 and D52 and assayed by ELISA for their antibodycontent. The results are expressed in log10 of the titer ofanti-lysozyme antibody (FIG. 13A) and anti-KLH antibody (FIG. 13B).

Three antigen injections were performed i.p. at days 0, 21 and 42. Thelysozyme-microparticles gave very good antibody responses while noantibody response was induced by the LH microparticles. Thesemicroparticles moreover very efficiently stimulated T responses.

One of the differences between LH and lysozyme is their molecularweights (14500 for lysozyme and 71000 for LH).

At equal concentrations of coupled antigen, the density of LH moleculeson the beads is thus about 5 times lower. This could explain the absenceof stimulation of antibody responses if these are due to T-independentdirect stimulation by the antigen present at high density on themicroparticles.

2. Hemoglobin and ovalbumin

Mice were immunized with soluble antigen in alum adjuvant or with thesame antigen in particulate form, in the absence of adjuvant. Antibodyappearance was then monitored over several weeks.

In the case of hemoglobin (Hb), the mice were immunized with 100 μg ofprotein or 10⁹ beads coupled with the protein at different densities(2.10⁴ and 2.10⁵ molecules/μm²). The beads carrying ovalbumin (OVA) weretested at two densities, 7.10³ and 7.10⁴ molecules/μm².

An initial injection was carried out, followed by two more injections onthe 21st and 40th days. Serums were taken at the 20th day, the 31st day,the 41st day and the 52nd day, then assayed by ELISA for their IgGantibody levels. The results are expressed in log of the antibody titer.

The results in FIG. 14 show that hemoglobin coupled to beads did notinduce an antibody response. For ovalbumin (FIG. 15) antibodies weredetectable after several injections if the antigen was coupled at highdensity, but these responses were weak. These results show that proteinsof high molecular weight such as hemoglobin are not able to induce anantibody response, even if they are coupled at a high density on thebeads.

These results, similar to those obtained with lysozyme and limulushemocyanin, confirm that beads carrying high-molecular-weight proteinsinduce T-proliferative responses in the absence of any antibodyproduction.

Likewise, proteins of low or medium molecular weight (less than 50 000)can induce the appearance of antibodies if they are coupled to the beadsat high densities.

3. Synthetic peptides

The peptides pre-S:TB (120-145) and pre-S:B corresponding to theportions of the HBS antigen containing respectively a T epitope and a Bepitope or only the B epitope were covalently coupled to 1 μm beads withglutaraldehyde (B-pre-S:TB and B-pre-S:B).

The antibody response induced by these beads was compared with thatinduced by 10 μg of soluble pre-S:TB peptide in alum adjuvant.

The results in FIG. 16 show that the beads coupled to the TB peptide,containing a T epitope and a B epitope, induced strong antibodyresponses, which confirms that antigens of low molecular weight coupledto beads are able to induce an antibody reaction in the absence ofadjuvant. It may be noted that these responses are as good as thoseobtained with the free peptide in the presence of alum adjuvant.

EXAMPLE 4

Effect of the lysozyme density on the microparticle surface on theirimmunogenicity

The materials and methods used were similar to those for Example 2.

The immunogenicity of beads coupled to lysozyme with different numbersof molecules on their surface was tested in experiments illustrated inFIGS. 17 and 18.

For FIG. 17A, BALB/c mice were immunized by subcutaneous injection of100 μg of lysozyme in CFA. After 14 days, the inguinal ganglions wereremoved and the cells tested in vitro against beads carrying differentdensities of lysozyme (from 1100 to 950 000 molecules of lysozyme on 1μm diameter beads). The results are expressed in cpm corrected for thevalue obtained without antigen.

For FIG. 17B, BALB/c mice were immunized by subcutaneous injection atthe base of the tail with soluble lysozyme with adjuvant (CFA) or 10⁹beads carrying different densities of lysozyme without adjuvant.

After 14 days, the inguinal ganglions were removed and the proliferativeresponse of these cells was tested in vitro against differentconcentrations of lysozyme or beads. The results are expressed in cpmcorrected for the value obtained without antigen.

The proliferation of ganglion cells originating from animals immunizedby soluble lysozyme in CFA was tested in vitro after stimulation bydifferent lysozyme-microparticles. The proliferative response of thesecells increased as the density of lysozyme on the microparticle surfaceincreased. No proliferation of the ganglion cells was obtained afterstimulation by microparticles with a density of 1100 lysozyme moleculesper microparticle (FIG. 17A).

In the experiment shown in FIG. 17B, the immunogenicity of thesemicroparticles was tested in vivo. BALB/c mice were immunized by thedifferent microparticles, without adjuvant, and the ganglion cells ofthese animals were stimulated in vitro by different concentrations ofsoluble lysozyme.

The proliferation of ganglion cells originating from animals immunizedby microparticles coupled to lysozyme at high density (950 000 and 210000) was high, and comparable to the response of cells sensitized by 100μg of lysozyme in CFA. After immunization by microparticles carrying amedium density of lysozyme (45 000), the cells proliferated in responseto lysozyme in vitro at concentrations from 10⁻¹ μg/ml. Lower-densitymicroparticles did not sensitize T cells in vivo, since no proliferationwas observed in the presence of lysozyme, even at high concentration(FIG. 17B).

It should be noted that 10⁹ microparticles coupled to lysozyme at highdensity correspond to 23 μg (1-950 000-G) and 5 μg (1-210 000-G) ofcoupled lysozyme, nevertheless the cell proliferation was as high asthat after injection with 100 μg of lysozyme in CFA.

For FIG. 18, BALB/c mice were immunized by subcutaneous injection oflysozyme with adjuvant (CFA) or of 10⁹ microparticles carrying differentdensities of lysozyme (950 000; 210 000, 45 000 and 1100 moleculesrespectively on a 1 μm diameter microparticle).

After 14 days, serums were taken and assayed by ELISA for their level ofanti-lysozyme antibody. The results are expressed in log10 of theantibody titer.

The humoral response of the mice immunized by these microparticles withdifferent lysozyme densities were studied. Injection of 100 μg oflysozyme in CFA induced a high level of anti-lysozyme antibody (FIG.18). Fourteen days after immunization, the beads coupled to the highestdensity of lysozyme (950 000) had induced significant antibodyproduction, while beads of lower density had not stimulated theinduction of a significant anti-lysozyme antibody response. Inparticular it should be noted that the beads with density 210 000, whichhad induced an excellent specific proliferation of the ganglion cells,did not stimulate antibody production. These results show that T cellproliferation is induced with lysozyme densities of between 45 000 and950 000 molecules per microparticle, while antibody production requiresa high density of protein coupled to the microparticles.

Within the meaning of the present description, the expression"microparticles" refers to particles which may have various geometricand spatial configurations. In practice, they are preferentiallymicrospheres or beads, such as are obtained by conventional polymermanufacturing techniques.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 5    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13    (B) TYPE: AMINO ACID    (C) STRANDEDNESS: UNKNOWN    (D) TOPOLOGY: UNKNOWN    (ii) MOLECULE TYPE: PEPTIDE    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetGlnTrpAsnSerThrThrPheHisGlnThrLeu    510    Gln    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14    (B) TYPE: AMINO ACID    (C) STRANDEDNESS: UNKNOWN    (D) TOPOLOGY: UNKNOWN    (ii) MOLECULE TYPE: PEPTIDE    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GlnAspProArgValArgGlyLeuTyrPheProAla    510    GlyGly    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13    (B) TYPE: AMINO ACID    (C) STRANDEDNESS: UNKNOWN    (D) TOPOLOGY: UNKNOWN    (ii) MOLECULE TYPE: PEPTIDE    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    LysLeuPheAlaValTrpLysIleThrTyrLysAsp    510    Thr    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11    (B) TYPE: AMINO ACID    (C) STRANDEDNESS: UNKNOWN    (D) TOPOLOGY: UNKNOWN    (ii) MOLECULE TYPE: PEPTIDE    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    AspAsnProAlaSerThrThrAsnLysAspLys    510    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40    (B) TYPE: AMINO ACID    (C) STRANDEDNESS: UNKNOWN    (D) TOPOLOGY: UNKNOWN    (ii) MOLECULE TYPE: PEPTIDE    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    IleAsnCysThrArgProAsnAsnAsnThrArg    510    LysSerIleArgIleGlnArgGlyProGlyArg    1520    AlaPheValThrIleGlyLysIleGlyAsnMet    2530    ArgGlnAlaHisCysAsnIle    3540    __________________________________________________________________________

We claim:
 1. A method of inducing an immune response in warm-bloodedanimals comprising administering to warm-blooded animals an immuneresponse inducing amount of synthetic biocompatible microparticleshaving an average diameter between about 0.25 μm and 1.5 μm and carryingon their surface at least one covalently bonded protein, each carryingat least one epitope to induce an humoral or cellular immune response,the density of the protein(s) on the microparticle surfaces beingadjusted to comprise between 10⁴ to 5.10⁵ molecules per μm² and todirect the said immune response towards the induction of cellular and/orhumoral response.
 2. The method of claim 1 of inducing an immuneresponse in warm-blooded animals comprising administering towarm-blooded animals an immune inducing amount of syntheticmicroparticles having an average diameter between about 0.25 μm and 1.5μm and carrying on their surface at least one covalently bonded protein,each carrying at least one epitope to induce an immune response, thedensity being comprised between 10⁴ to 5.10⁴ molecules per μm² and themolecular weight of the protein(s) on the microparticle surfaces beingadjusted to direct a T cell immune response.
 3. The method of claim 1,for the induction of cellular and/or humoral responses, characterized inthat the microparticles have a density for each of the proteins carryingan epitope of a minimum of 10⁵ molecules/μm² and preferentially 5.10⁵molecules/μm².
 4. The method of claim 1, for the induction of a mainlycellular response, characterized in that the microparticles have adensity for each of the proteins carrying an epitope of between 10⁴ and5.10⁴ molecules/μm².
 5. The method of claim 1, for the induction of amainly cellular response, characterized in that the microparticles carryon their surface proteins having molecular weights greater than 50 kD.6. The method of claim 1, characterized in that the microparticles havean average diameter of between about 0.25 μm and about 1.5 μm, andpreferentially of 1 μm.
 7. The method of claim 1, characterized in thatthe bond is formed by reaction between the NH₂ and/or CO groups of theproteins and the material making up the microparticle.
 8. The method ofclaim 1, characterized in that the bond between the proteins and thematerial making up the microparticle is covalent and formed with orwithout a bridging reagent.
 9. The method of claim 8, characterized inthat the bridging reagent is glutaraldehyde or carbodiimide.
 10. Themethod of claim 1, characterized in that said microparticles arebiocompatible polymers.
 11. The method of claim 10, characterized inthat said polymer is polyacrolein or polystyrene or lactic acid polymersor copolymers of lactic and glycolic acids.
 12. The method of claim 1,characterized in that the microparticles carry on their surfacemolecules able to activate the immune system.
 13. Process for theproduction of microparticles whose immune response is either mainlyhumoral or mainly cellular, said process being characterized in that atleast one protein carrying one or more epitopes or peptides containing Tor B epitopes alone or a combination of the two are covalently fixed tosynthetic polymer microparticles, the density of the protein fixed tothe surface being varied according to the humoral or cellular responserequired.
 14. Process according to claim 13, characterized in thatmicroparticles as defined in claim 1 are used.
 15. Syntheticbiocompatible microparticles having an average diameter between about0.25 μm and 1.5 μm and carrying on their surface at least one covalentlybonded protein, each carrying at least one epitope to induce an humoralor cellular immune response, the density of the protein(s) on themicroparticle surfaces being adjusted to comprise between 10⁴ to 5.10⁵molecules per μm² and to direct the said immune response towards theinduction of cellular and/or humoral response.
 16. Microparticles ofclaim 15 wherein the protein is selected from the group consisting ofhemoglobin, ovalbumin and lysozyme.
 17. Microparticles of claim 15,having an average diameter of 1 μm.
 18. Microparticles of claim 15,characterized in that the bond is formed by reaction between the NH₂and/or CO groups of the proteins and the material making up themicroparticle.
 19. Microparticles of claim 15 wherein the bond betweenthe proteins and the polymer microparticle is formed by use of abridging reagent.
 20. Microparticle according to claim 19, characterizedin that the bridging reagent is glutaraldehyde, or carbodiimide. 21.Microparticle according to one of claim 15, characterized in that it iscomposed of a biocompatible polymer.
 22. Microparticle according toclaim 21, characterized in that said polymer is poly(acrolein) orpolystyrene, a lactic acid polymer or a copolymer of lactic and glycolicacids.
 23. Microparticle according to claim 17, characterized in that itcarries on its surface molecules able to activate the immune system. 24.Microparticle according to claim 17, characterized in that said proteincomprises the B epitope from the pre-S₂ region of the HBs antigen of theviral hepatitis virus.
 25. Microparticle according to claim 17,characterized in that said protein comprises the B epitope of the VP1protein of the poliomyelitis virus.
 26. Microparticle according to claim17, characterized in that said protein comprises the B epitope of thegp120 protein of the HIV-1 virus.
 27. Pharmaceutical compositioncharacterized in that it comprises microparticles according to claim 17,in combination with pharmaceutically compatible diluents or adjuvants.